MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that control gene expression by directing their target mRNAs for degradation and/or posttranscriptional repression. Abnormal expression of miRNAs is thought to contribute to the development and progression of breast (and other) cancer. However, we are not aware of any studies in humans that have prospectively examined miRNA expression in relation to risk of (breast) cancer development. Therefore, we propose to conduct nested case-control studies to first identify and then validate miRNAs that are related to the risk of breast cancer development. miRNAs are conserved intact in formalin-fixed paraffin-embedded (FFPE) tissue. Hence, our work will be undertaken in an ongoing cohort study of 15,395 women who had a biopsy for benign breast disease (BBD) between 1971 and 2006, for whom FFPE breast tissue samples are accessible, and who are being followed up to ascertain the occurrence of breast cancer subsequent to BBD. Cases (n=625) will be women with BBD who subsequently developed breast cancer, and one control will be matched individually to each case. In the identification phase, RNA extracted from FFPE BBD tissue blocks of 415 case-control pairs will be analyzed using the Illumina MicroRNA Expression Profiling Assay to identify the top 70 miRNAs that are differentially expressed between cases and controls. These candidate miRNAs will be confirmed using qRT-PCR, and the 20 miRNAs that show the strongest correlation between the Illumina and qRT-PCR results will then be tested for case-control differences using qRT-PCR in a separate nested case-control study involving 210 case- control pairs. In additional analyses, we will examine the association between miRNA expression and risk of breast cancer classified by estrogen/progesterone receptor status, and, for a random sample of cases, we will compare miRNA expression in benign breast disease tissue with that in the corresponding breast cancer tissue. Our ultimate goal is to enhance approaches both to the identification of women at risk of breast cancer and to the clinical management of such women. The proposed project has translational potential in that it might facilitate progress towards accomplishment of our long-term goals.